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MITOCHONDRIAL DNA MARKER BASED PHYLOGENETIC ANALYSIS OF PAKISTANI NILGAI (Boselaphus tragocamelus).

Byline: G. Abbas, A. Nadeem, M. E. Babar, T. Hussain, N. Aslam, W. Shehzad, M. Tayyab and M. Javed

ABSTRACT

The Nilgai or Blue Bull (Boselaphus tragocamelus) is the largest Asian antelope which is endemic to Nepal, Pakistan and Indian regions, where these animals are found in packs. Due to rampant hunting, deforestation and habitat degradation, this vulnerable specie is near threatened in many parts of the world. There is need of conservation efforts both at genomic and geographic level. The present study was designed for genomic characterization of Boselaphus tragocamelus in Pakistan by using phylogenetic analysis. Samples from Boselaphus tragocamelus were collected from different parks, zoos and natural habitats. DNA was extracted by standard inorganic extraction method. Primer3 software was used for primer designing targeting Mitochondrial specie identification markers such as d-loop, cytochrome-b and cytochrome-c. After amplification, PCR product was sequenced. Bioinformatics tools were applied for identification of polymorphic loci. Allelic frequency of each locus was calculated.

Multidimensional scaling plot illustrated low level of generic diversity among individuals. Phylogenetic analysis revealed conserved neighbouring pattern among different individuals as they shared common ancestry. This is the first report on genomic characterization of Nilgai from Pakistan. The information of selected species of deer is prerequisite for designing effective strategy in future effective practices fo r conservation. However further genomic investigations should be carried out at a larger scale.

Keywords: Mitochondrial cytochrome-b, cytochrome-c, d-loop, Phylogenetic, Pakistani nilgai.

INTRODUCTION

The largest of the Asian antelopes, the Nilgai (Boselaphus tragocamelus) is a bovid that was defined for the first time in 1766 by Pallas. The body of female Nilgai is yellow-brown in color. In males, yellow-brown color steadily changes into blue-grey when they approach the mature age. Their nape and back have erectable mane. A "hair pennant" is visible in the middle of the underside of the neck. White patterns are visible as cheek spots, along edges of the lips and as a throat bib. Underside of body has a thin white stripe which expands in size as it proceeds towards backside. Legs are slim that provide support to stocky body. The body slopes downwards from front towards back. The head is long and slim. Horns of males Blue bull are 20 to 25 cm long. Horns are straight and tilted forward slightly. Favorite habitats of Nilgai are woodlands areas and grassy steppe. Abodes of Nilgai in Pakistan are pure desert areas of Cholistan and Thar in Punjab and Sind respectively.

Nowadays, this Antelope is naturally found in a narrow strip of land along Indian border in the eastern part of Pakistan. They are not found in herds in this area. Presence of Nilgai in Changa Manga Plantation, near Lahore, has also been reported also. According to Sind Wildlife Department, 220 Nilgai were found in Tharparkar district in 1999 (Maan and Chaudhry 2001).

For phylogenetic studies and species identification of different animals, mitochondrial and nuclear DNA sequences have been used frequently in the recent years (Hsieh et al., 2001). Molecular phylogenetic and diversity analysis of Pakistani buffalo (Saif et al., 2012; Hussian et al., 2009; 2013a: 2015), goat (Hussain et al., 2013b), sheep (Babar et al, 2014; Ahmedet al., 2014), chicken (Babar et al., 2012), camel (Babar et al., 2015) and dog (Tahir et al., 2015) have already been reported but information on wildlife species is inadequate. In this context, present research was planned for genomic characterization by sequencing of mitochondrial cytochrome-b, cytochrome-c and d-loop regions of Pakistani nilgai (Boselaphus tragocamelus).

MATERIALS AND METHODS

Texonomic Specie and Sampling Strategy: Nilgai (Boselaphus tragocamelus) specie of animal from Bovidae family was selected for this study. Natural habitats of the specie in Pakistan and sampling areas have been depicted in Fig-1. Unrelated animals of the mentioned Bovidae family were selected for sampling. A total of 25 fecal and blood samples were collected. The selection was purely based upon phenotypic characteristics. Sampling was done from natural habitats, zoos, parks, wildlife reserves and captive breeding centers at different places of Pakistan. Sampling details have been provided in Table-1.

Genome Extraction and Purification: Fecal samples were collected using disposable plastic gloves and and stored this mass in 95% ethanol, at room temperature, using a polypropylene bottle. Fecal DNA was extracted as described by Zhang et al., (2006) and quantified by Nanodrop (Thermoscientific, Wilmington USA).

Blood samples were collcted from animals in captivity, in EDTA coated vacutainers and stored at -200C. DNA was extracted by using standard inorganic extraction protocol reported by Maryam et al., (2012) and quantified by Nanodrop.

Primer Designing: Reference sequences of complete mitochondrial genome including cytochrome-b, cytochrome-c and d loop regions for Boselaphus tragocamelus (Accession No.NC_020614) was retrieved from NCBI (www.ncbi.nlm.nih.gov). Primers were designed for complete amplification of three loci. Primers were designed using the primer blast of NCBI (www.ncbi.nlm.nih.gov) and synthesized at Genelink, USA.

PCR Amplification and DNA Sequencing: PCR was performed using Bio-Rad Thermocycler. PCR products of all the samples along with 1 kb ladder were run on 1.2% agarose gel at 100 volts for 35 minutes, to visualize the bands of amplified products. Precipitated PCR products were sequenced using dye labelled dideoxy terminatore sequencing using ABI Genetic Analyzer 3130 XL (Applied Biosystem Inc., Foster city, CA, USA).

Bioinformatic and Statistical Analysis: Alignments of sequences were done with the help of Blast 2 Sequences and Clustal W (Thompson et al., 1994). Maximum Parsimony Tree and Bayesian Phylogenetic tree was constructed by using MEGA2 (Kumar et al., 2001). Further analysis was done by using Bioconductor in R (Gentleman et al., 1999) to draw Multidimensional scaling plot and genetic variation plot for all three genes to evaluate the diversity score among individuals.

RESULTS

Phylogenetic analysis of Boselaphus tragocamelus using cytochrome-b Gene: A total of thirteen polymorphisms were identified in cytochrome-b gene of Boselaphus tragocamelus as given in Table 2. Allele frequency was calculated. As no heterozygosity was observed so genotypic frequency was found same as allele frequency. Multidimensional scaling (MDS) plot was generated by using 'R'. Plot was figured out by using 1st and 2nd dimensional transformations showing symmetrical variation of genetic distance values in MDS plot. Sequences were analyzed to get evolutionary distance matrix. That matrix was then utilized to plot MDS as shown in Figure 2. Computational model (BH87) was used to generate distance profile for cytochrome-b sequence of Boselaphus tragocamelus. Genetic variation plot was created by computational model for Boselaphus tragocamelus of various regional origins. Figure 3 was generated by using the genetic distance dataset of mitochondrial genomic region of cytochrome-b.

The Neighbor joining phylogenetic tree was constructed by using maximum likelihood method implemented in a desktop application named as MEGA 6. Boselaphus tragocamelus was the target specie. DNA sequences of cytochrome-b gene of Boselaphus tragocamelus were processed for the phylogenetic analysis. Target specie is encircled in Figure 4.

Cytochrome-c Gene based analysis: A total of sixteen polymorphisms were identified in cytochrome-c gene of Boselaphus tragocamelus as given in Table 3. No heterozygosity was observed. Allele frequency "1" represents that all samples were monomorphic. MDS plot (figure 5), pair wise evolutionary distance (figure 6) and phylogenetic tree (figure 7) was constructed as described earlier.

Mitochondrial d-loop region based analysis: A total of seventeen polymorphisms were identified in mitochondrial d-loop region of Boselaphus tragocamelus as given in Table 4. No heterozygosty was observed. MDS plot (Figure 8), pair wise evolutionary Distance (Figure 9) and phylogenetic tree (Figure 10) was also contructed.

Mitochondrial cytochrome-b, cytochrome-c and d-loop region (three genes combined sequence) based analysis: Sequence of three genes (cytochrome-b, cytochrome-c and d-loop) were combined and multidimensional scaling plot was generated. Plot was figured out by using 1st and 2nd dimensional transformations showing symmetrical variation of genetic distance values in MDS plot (Figure 11). pair wise Evolutionary distance (Table 5) and estimates of evolutionary divergence between sequences (Figure 12) were calculated. Phylogenetic tree was constructed by using maximum likelihood method as described earlier. DNA sequences of cytochrome-b, cytochrome-c and d-loop region (combined sequence) of animals under study were routed for phylogenetic analysis (Figure 13).

Table 1. Sampling details of Boselaphus tragocamelusa samples used in this study.

###Sr. No.###Samples###Source###Google Coordinates

###1###BTa5 BT17###Bahawalpur Zoo###29deg24'8.7"N 71deg40'54.5"E

###2###BT7, BT23###Bahria Town Lahore###31deg18'51.5"N 74deg12'11.7"E

###3###BT10, BT19, BT20###Bahria Town Rawalpindi###33deg29'45.2"N 73deg6'20.3"E

###4###BT8###Basti Bahadurpur Multan###30deg15'27.9"N 71deg29'48.2"E

###5###BT2###Changa Manga Kasur###31deg5'19.3"N 73deg57'44.7"E

###6###BT4###Charagh Abad, T T Sing###31deg20'6.3"N 72deg46'2.4"E

###7###BT15 BT22###Gatwala Wildlife Breeding Centre, Faisalabad###31deg28'42.7"N 73deg12'36.7"E

###8###BT16###Lahore safari park, Lahore###31deg22'53.9"N 74deg12'41.6"E

###9###BT18###Lahore Zoo, Lahore###31deg33'22.7"N 74deg19'34.0"E

###10###BT9, BT11###Lal Suhanra National Park Bahawalpur###29deg19'1.4"N 71deg54'16.4"E

###11###BT14, BT21###Lohi Bher Wildlife Park Rawalpindi###33deg57'49.5"N 73deg11'93.1"E

###12###BT24###Indo-Pak border, Bahawalnagar###29deg59'57.1"N 73deg15'31.8"E

###13###BT25 BT3###Peerowal Khanewal###30deg20'22.7"N 72deg2'2.4"E

###14###BT1 BT6###Head Balloke Raavi River###31deg11'25.9"N 73deg52'32.6"E

###15###BT13###Vehari Wildlife Park Vehari###30deg2'14.7"N 72deg21'2.6"E

###16###BT12###Wildlife Park Kamalia, T T Sing###30deg42'52.9"N 72deg40'25.9"E

Table 2. Polymorphisms in cytochrome-b gene of Boselaphus tragocamelus.

###No.###Base Positiona###Change in Nucleotide###Allele Frequency

###(Wild to Mutant)###A###B

###1###14180###A C###0###1b

###2###15286###C T###0###1b

###3###15302###T G###0###1b

###4###14293###CT###0.92###0.08

###5###14635###CT###0.64###0.36

###6###15121###TC###0.92###0.08

###7###15134###CT###0.92###0.08

###8###15135###TC###0.92###0.08

###9###15138###GA###0.92###0.08

###10###15154###CT###0.64###0.36

###11###15179###GA###0.80###0.20

###12###15252###AG###0.84###0.16

###13###15290###TC###0.60###0.40

Table 3. Polymorphisms in cytochrome-c gene of Boselaphus tragocamelus.

###No.###Base Positiona###Change in Nucleotide###Allele Frequency

###(Wild to Mutant)###A###B

###1###5497###T A###0###1

###2###6260###G A###0###1

###3###6395###G A###0###1

###4###6668###G A###0###1

###5###6707###C T###0###1

###6###5469###AG###0.80###0.20

###7###5472###CT###0.80###0.20

###8###5493###AG###0.80###0.20

###9###5496###GA###0.80###0.20

###10###5867###CT###0.64###0.36

###11###5871###AG###0.64###0.36

###12###5937###TC###0.64###0.36

###13###6260###GA###0.88###0.12

###14###6395###GA###0.88###0.12

###15###6668###GA###0.88###0.12

###16###6818###CT###0.68###0.32

Table 4. Polymorphisms in d-loop region of Boselaphus tragocamelus.

###No.###Base Positiona###Change in Nucleotide###Allele Frequency

###(Wild to Mutant)###A###B

###1###15684###T C###0###1

###2###15834###C T###0###1

###3###16284###C G###0###1

###4###15556###GA###0.68###0.32

###5###15606###TC###0.76###0.24

###6###15647###AG###0.88###0.12

###7###15662###TC###0.88###0.12

###8###15663###CT###0.88###0.12

###9###15685###CT###0.64###0.36

###10###15705###TC###0.88###0.12

###11###15713###GA###0.88###0.12

###12###15835###TC###0.64###0.36

###13###16085###TC###0.88###0.12

###14###16241###AG###0.80###0.20

###15###16283###AT###0.80###0.20

###16###16285###CG###0.64###0.36

###17###16288###AG

###0.80###0.20

Table 5. Cytochrome-b, cytochrome-c and d-loop region based evolutionary analysis of Boselaphus tragocamelus.

[###1###2###3###4###5###6###7###8###9###10###11###12###13###14###15###16###17###18###19###20###21###22###23###24###25

[ 1]

[ 2]###0.00365

[ 3]###0.00169###0.00253

[ 4]###0.00478###0.00281###0.00365

[ 5]###0.00309###0.00169###0.00197###0.00337

[ 6]###0.00197###0.00281###0.00084###0.00393###0.00225

[ 7]###0.00112###0.00478###0.00281###0.0059###0.00422###0.00309

[ 8]###0.0045###0.00309###0.00393###0.0059###0.00309###0.00365###0.00393

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[13]###0.0045###0.00422###0.00337###0.00197###0.00309###0.00365###0.00393###0.00393###0.00422###0.0045###0.00309###0.00309

[14]###0.00478###0.00393###0.00422###0.00675###0.00337###0.00337###0.00478###0.00365###0.00169###0.00365###0.00337###0.00337###0.00534

[15]###0.00365###0.00056###0.00309###0.00337###0.00225###0.00281###0.00422###0.00309###0.00506###0.00646###0.00225###0.00393###0.00478###0.00393

[16]###0.00253###0.00393###0.00141###0.00506###0.00337###0.00169###0.00365###0.00253###0.00393###0.00646###0.00337###0.00225###0.00478###0.00506###0.00393

[17]###0.00141###0.00225###0.00309###0.00337###0.00393###0.00281###0.00253###0.00478###0.00506###0.00422###0.00393###0.00393###0.00478###0.00562###0.00225###0.00393

[18]###0.00253###0.00112###0.00141###0.00393###0.00056###0.00169###0.00365###0.00253###0.00337###0.00534###0.00056###0.00281###0.00365###0.00281###0.00169###0.00281###0.00337

[19]###0.0045###0.00084###0.00337###0.00197###0.00141###0.00365###0.00562###0.00393###0.00534###0.00506###0.00141###0.00478###0.00393###0.00478###0.00141###0.00478###0.00309###0.00197

[20]###0.00197###0.00225###0.00028###0.00337###0.00169###0.00056###0.00309###0.00365###0.00225###0.00478###0.00169###0.00169###0.00309###0.00393###0.00281###0.00169###0.00281###0.00112###0.00309

[21]###0.00365###0.00393###0.00253###0.00112###0.00225###0.00281###0.00478###0.00534###0.0045###0.00478###0.00225###0.00393###0.00141###0.00562###0.0045###0.00393###0.0045###0.00281###0.00309###0.00225

[22]###0.00365###0.00281###0.00253###0.0045###0.00112###0.00337###0.00309###0.00253###0.00393###0.00478###0.00112###0.00225###0.00253###0.00337###0.00337###0.00393###0.00506###0.00169###0.00253###0.00281###0.00337

[23]###0.00309###0.00337###0.00197###0.0045###0.00337###0.00112###0.00253###0.00253###0.00281###0.00478###0.00337###0.00056###0.00253###0.00337###0.00337###0.00281###0.00337###0.00281###0.00422###0.00169###0.00393###0.00281

[24]###0.00112###0.00422###0.00225###0.00365###0.00253###0.00309###0.00225###0.00562###0.00478###0.00281###0.00253###0.00365###0.00393###0.0059###0.00478###0.00365###0.00253###0.00309###0.00337###0.00253###0.00253###0.00309###0.00422

[25]###0.16554###0.16582###0.1647###0.16554###0.1661###0.1647###0.16554###0.16695###0.1661###0.16835###0.1661###0.16498###0.16554###0.16695###0.16526###0.16554###0.16582###0.16554###0.16667###0.1647###0.16526###0.16639###0.16526###0.16667

[26]###0.00225###0.00309###0.00169###0.00422###0.00253###0.00141###0.00225###0.00281###0.00253###0.0045###0.00253###0.00141###0.00281###0.00309###0.00309###0.00253###0.00309###0.00197###0.00393###0.00141###0.00309###0.00253###0.00141###0.00337###0.1647

DISCUSSION

Conservation of genome sequences might play a significant role in species survival and ecosystem conservation in the future. For the purpose of collecting genetic information for on Boselaphus tragocamelus specie in Pakistan, mitochondrial cytochrome-b, cytochrome-c and d-loop regions were analysed to study the genetic diversity and used for construction of Phylogenetic tree (Giovambattista et al., 2001; Feral et a.l, 2006 and Mwacharo et a1., 2006). DNA sequence based techniques are helpful for evaluating genetic changeability between different populations (Haig; 1998). The wild animal's conservation is rare and depends on protection of genetic differences which is sign of difference in genomes (Crozier,1992; Lynch and Milligan, 1994). To assess the level of genetic variation, an 1139 bp fragment, in the cytochrome-b gene of the mitochondrial DNA, from 25 deer individuals was PCR-amplified and sequenced and analysed.

A total of thirteen variable sites were observed in cytochrome-b gene of Boselaphus tragocamelus. Out of these, three variations were found monomorphic for mutant allele. Remaining animals were also homozygous both for wild and mutant allele. The variable sites were comprised of 10 transitions and three transversions. Allele frequency of all variations was calculated and very low frequency of mutant allele was observed. As no heterozygous individuals were found so allelic frequency and genotypic frequency was the same. The average heterozygosity values for endangered and non-endangered populations are often lower and higher values, respectively (Frankham et al., 2002). So, our results illustrate distribution of specie inclined more towards endangered, which is alarming and demands immediate measures for its conservation. In some other related studies as well, overall genotypic frequency has been found to be homozygous as Nielsen et al., (2008), Qureshi et al., (2004) and Dellicour et al., (2011).

Pepin et al. (1995) reported that conservation of genome nucleotide sequence occurs in Nilgai (Boselaphus tragocamelus). Gallagher et al. (1998) established that Nilgai was karyotypically derivative to genus of Bovinae; various resultant chromosomal conditions familiar to B. tragocamelus, the buffalo from Africa and various Tragelaphini can be convergent. Nucleotide sequence of different genes such as interferon2, Toll like receptor3 and prion protein suggest lineage share of this species with buffalo, kudu and cattle (Das et al. 2006: Dhara et al. 2007: Seabury et al., 2004). Similar outcomes have been illustrated in the phylogenetic analysis (Fig-13). Genetic variation information is prerequisite for future conservation strategy (Crandall et al., 2000). It is recommended that further genomic investigations should be carried out at a larger scale.

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Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Jun 30, 2017
Words:4606
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